Dynactin is a highly conserved, ubiquitous protein complex that contributes to cytoplasmic dyneinbased motility and other fundamental cellular behaviors. Its composition and overall structural features are well-defined, but detailed structural information has not yet emerged due to the lack of a suitable recombinant expression system. EM analysis of dynactin isolated from tissue sources has provided good resolution, but the most promising avenue to obtaining high resolution structural information is subcomplexes that correspond to defined domains of the dynactin molecule. Such subcomplexes are also extremely useful for biochemical studies aimed at defining interactions within the dynactin molecule and identifying and characterizing binding partners. In this proposal, I describe experiments on two different dynactin subcomplexes, the shoulder/sidearm and the pointed end complex, which mediate motor binding and cargo specification, respectively. Interactions between the shoulder/sidearm, dynein and the plus end-directed motor, kinesin-2, will also be evaluated. The experiments described in Specific Aim 1 are intended to provide new insights into the structure and organization of the dynactin shoulder/sidearm, which is composed of p150Glued, dynamitin and p24. The question of whether shoulder/sidearm stability is phosphoregulated will also be explored. At the opposite end of the dynactin molecule from shoulder/sidearm is the pointed end complex, which is comprised of the subunits p62, Arp11, p27 and p25. Pointed end complex shows compositional and structural variability that appears to be due to dynamic interactions between p27 and p25 and the rest of the dynactin molecule. We find that p27 is a substrate for the mitotic kinase, Cdk1, suggesting a novel mechanism for regulating p27 interactions. We propose that p27 phosphorylation provides a means for altering dynactin targeting and activity across the cell cycle. In Specific Aim 2, we will explore the structure of the p27/p25 heterodimer and evaluate how cell cycle phosphorylation impacts p27 behavior in vivo and in vitro. Dynactin is best known for its roles in cytoplasmic dynein-based motility, but it also contributes to the function of kinesin-2. Working together, dynein, dynactin and kinesin-2 power the bidirectional movement of late endosomes and pigment granules. In Specific Aim 3, we will analyze the interplay among these proteins using in vitro interaction and motility assays. The motility assays will also be used to evaluate the effect impact of the kinases whose functions will be evaluated in Specific Aims 1 and 2.